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Disclaimer Clinicopathologic features and molecular characteristics of glucose metabolism contributing to 18F-fluorodeoxyglucose uptake in gastrointestinal stromal tumor Min-Hee Cho Department of Medical Science The Graduate School, Yonsei University Clinicopathologic features and molecular characteristics of glucose metabolism contributing to 18F-fluorodeoxyglucose uptake in gastrointestinal stromal tumor Directed by Professor Hoguen Kim The Master's Thesis submitted to the Department of Medical Science, the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Master of Medical Science Min-Hee Cho December 2015 This certifies that the Master's Thesis of Min-Hee Cho is approved. _________________________________ Thesis Supervisor: Hoguen Kim _________________________________ Thesis Committee Member#1: Kyung-Sup Kim _________________________________ Thesis Committee Member#2: Jae-Ho Cheong The Graduate School Yonsei University December 2015 ACKNOWLEDGEMENTS 아직도 모르는 것이 너무 많은데 어느덧 졸업을 앞두고 있으니, 기쁘지만 걱정스러운 마음이 더 큽니다. 시행착오 속에서 제가 학위논문을 마무리할 수 있었던 이유는, 학위기간 중 저를 응원해주고 도와주셨던 분들 덕분이라고 생각합니다. 먼저 저를 제자로 받아주시고, 많은 성과를 내지 못했는데 끝까지 기다려주시고 지도해주신 김호근 교수님께 진심으로 감사 드립니다. 더 좋은 학위논문이 될 수 있도록 조언해 주신 김경섭 교수님, 정재호 교수님께도 깊은 감사를 드립니다. 또한 입학할 때 격려해주시고 용기 북돋아 주신 이진아 교수님, 실험을 배워볼 수 있도록 기회를 주셨던 황기철 교수님, 창연오빠, 세연언니 모두 감사 드립니다. 항상 옆에서 응원해줬던 다솜이, 지영이언니, 시정이언니, 복경이, 광현오빠, 용우오빠 너무 고맙고, 다른 실험실이지만 이것저것 많이 알려주고 도움 준 이나언니도 너무 고맙습니다. 그리고 실험하면서 여러 코멘트 해주고 도와준 실험실 선배,후배님들 감사 드립니다. 마지막으로, 몸도 많이 편찮으신데 부족한 딸 여기까지 공부시켜주신 엄마, 아빠 감사하고 사랑한다는 말 전합니다. 제 곁에 있는 것만으로 큰 힘이 됩니다. 졸업은 또 다른 시작이니 새롭게 시작하는 저를 지금처럼 응원해주시고 지켜봐 주시길 바랍니다. 감사합니다. 2015년 12월 저자 조민희 올림 TABLE OF CONTENTS ABSTRACT ····································································· 1 I. INTRODUCTION ·························································· 3 II. MATERIALS AND METHODS ········································ 7 1. Patient selection ························································· 7 2. Mutational analysis ······················································ 7 3. PET/CT protocol and quantification ································· 8 4. Immunohistochemistry ················································· 9 5. Western blotting ························································ 10 6. Quantitative reverse transcription-polymerase chain reaction ··· 11 7. Hexokinase and lactate dehydrogenase activity assay ············ 12 8. Statistical analysis ····················································· 13 III. RESULTS ································································· 14 1. Clinicopathologic characteristics and 18F-FDG uptake of 40 GISTs ··········································································· 14 2. Correlation between 18F-FDG uptake and tumor risk grade ··········································································· 22 3. Overexpression of GLUT1 and HK1 in GISTs according to tumor risk grade ······························································ 22 4. Enhanced expression of PKM2 and LDHA in high-risk GISTs ··········································································· 29 IV. DISCUSSION ····························································· 34 V. CONCLUSION ···························································· 37 REFERENCES ······························································· 38 ABSTRACT (IN KOREAN) ··············································· 43 PUBLICATION LIST ······················································· 46 LIST OF FIGURES Figure 1. Histologic features of GISTs and Representative cases showing 18F-FDG uptake intensity according to tumor risk grade ··········································18 Figure 2. Total HK activity in GISTs ···························· 23 Figure 3. GLUTs (GLUT1, 2, 3, and 4) and HKs (HK1 and 2) expression according to GIST risk grade, as assessed by quantitative RT-PCR ····················· 24 Figure 4. SUVmax correlated with GLUT1 and HK1 expression ····················································· 27 Figure 5. PKM2 and LDHA expression according to GIST risk grade, as assessed by quantitative RT-PCR ········ 29 Figure 6. SUVmax correlated with LDHA expression ······ 32 Figure 7. LDH activity in GISTs ···································· 33 LIST OF TABLES Table 1. Demographic and clinicopathologic features of 40 patients with GIST ···································· 15 Table 2. Relationships between GIST risk grade and 18F-FDG uptake and demographic and clinicopathologic factors··················································· 17 Table 3. Comparison of clinicopathologic categories between GISTs with KIT mutation ···························· 20 Table 4. Relationships between GLUT1 and HK1 immunohistochemical expression and GIST tumor risk grade ··············································· 26 Table 5. Relationships between GLUT1 and HK1 immunohistochemical expression and tumor size and mitotic count ····································· 28 Table 6. Relationships between PKM2 and LDHA immunohistochemical expression and GIST tumor risk grade ·············································· 31 ABSTRACT Clinicopathologic features and molecular characteristics of glucose metabolism contributing to 18F-fluorodeoxyglucose uptake in gastrointestinal stromal tumor Min-Hee Cho Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Hoguen Kim) Fluorine-18 fluorodeoxyglucose (18F-FDG) positron emission tomography–computed tomography (PET/CT) is useful in the preoperative diagnosis of gastrointestinal stromal tumors (GISTs); however, the molecular characteristics of glucose metabolism of GIST are unknown. We evaluated 18F-FDG uptake on preoperative PET/CT of 40 patients and analyzed the expression of glycolytic enzymes in resected GIST tissues by qRT-PCR, western blotting, and immunohistochemistry. Results of receiver operating 1 characteristic curve analysis showed that the maximum standardized uptake value (SUVmax) cut-off value of 4.99 had a sensitivity of 89.5%, specificity was 76.2%, and accuracy of 82.5% for identifying tumors with a high risk of malignancy. We found that 18F-FDG uptake correlated positively with tumor risk grade and expression levels of glucose transporter 1 (GLUT1), hexokinase 1 (HK1), and lactate dehydrogenase A (LDHA). Overexpression of GLUT1 and HK1 increased with higher tumor risk grade. In addition, overexpression of glycolytic enzymes M2 isoform of pyruvate kinase (PKM2) and LDHA was observed in GISTs, especially in high-risk tumors. These results indicate increased glycolysis in GISTs and suggest that upregulation of GLUT1, HK1, PKM2, and LDHA may play an important role in GIST tumorigenesis and may be useful in the preoperative prediction of malignant potential. ------------------------------------------------------------------------------------------------ Key words: gastrointestinal stromal tumors, 18F-FDG uptake, glucose metabolism, malignancy 2 Clinicopathologic features and molecular characteristics of glucose metabolism contributing to 18F-fluorodeoxyglucose uptake in gastrointestinal stromal tumor Min-Hee Cho Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Hoguen Kim) I. INTRODUCTION Gastrointestinal stromal tumors (GISTs), the most common non-epithelial neoplasms of the gastrointestinal (GI) tract, are defined as “KIT or PDGFRA mutation-driven mesenchymal tumors that can occur anywhere in the GI tract”.1 Preoperative diagnosis of GISTs and assessment of their malignant potential are difficult because most GISTs are located in the submucosa. Tumor grading is therefore based primarily on mitotic index and tumor diameter. 3 The clinical usefulness of 18F-fluorodeoxyglucose (FDG) positron emission tomography–computed tomography (PET/CT) has been demonstrated in tumor staging, treatment response assessment, and prognosis prediction in various tumors.2-6 This technique is also used in tumor staging and evaluation of targeted therapy response in GISTs.7-12 Many tumors depend on aerobic glycolysis for rapid growth beyond that supported by the vasculature. 18F-FDG is an analogue of glucose that allows noninvasive evaluation of the tumor’s glucose metabolism, which can predict treatment response and patient prognosis. 18F-FDG enters the cell through 18 glucose transporters (GLUT), is phosphorylated to F-FDG-6-PO4 by hexokinase (HK), and is then “trapped” in the cell as it is not further metabolized, allowing PET/CT acquisition 1 hour after 18F-FDG injection. Upregulation of GLUT and HK expression is associated with increased glucose metabolism and 18F-FDG uptake in tumors. For example, previous studies have shown that increased GLUT1 expression correlates with higher 18F-FDG uptake in lung and breast carcinomas.13-15 Although 18F-FDG PET/CT is useful for the preoperative diagnosis of GISTs, the detailed molecular mechanisms underlying glucose metabolism in these tumors and specific characteristics associated with tumor risk grade are not well 4 understood. Tumor cells consume large amounts of glucose and